Farzaneh Sabahi

Molecular epidemiology of hepatitis B virus in Iran

Hepatitis B virus (HBV) infection is a major cause of liver disease worldwide. Eight genotypes and 24 subgenotypes of HBV have been identified. The aim of this study was to determine the distribution of HBV genotypes, subgenotypes and subtypes, and to understand HBV genetic variability in the HBV genome circulating in Iranian provinces. Two hundred and forty-nine sera from HBV-infected patients living in 25 provinces of Iran were collected (2004-2007). A part of the HBV S/pol and whole BCP/C genes were amplified, sequenced and then subjected to phylogenetic, recombination and genetic variability analysis. Results revealed genotype D of HBV in all samples and subgenotypes D1 (98.52%), D2 (0.74%) and D3 (0.74%) among Iranian patients living in different provinces of Iran. Subtypes ayw2 (94.4%), ayw1 (2.8%), ayw3 (2%) and ayw4 (0.4%) were deduced, on the basis of HBV small surface antigen (HBsAg) amino acid sequences. The mean percentage intra-genotypic distance of S plus core regions was 2.8%; the mean percentage inter-genotypic distance of this region between Iranian strains and genotype D isolates was 3.1%; and this rate for other genotypes was 5.2-11.4%. Various rates of point mutations have been found within different HBV genes, e.g. HBsAg (17.2%), precore-G1896A (59.5%) and Basal core promoter (BCP) double mutations (49.2%), whereas no recombination was found. In conclusion, these results indicate that the only genotype circulating in the provinces of Iran is genotype D. There exist high genetic variabilities in the S/pol and BCP/C regions among the Iranian HBV isolates.

A novel adjuvant, the general opioid antagonist naloxone, elicits a robust cellular immune response for a DNA vaccine

While many adjuvants have been discovered and used in research, only a few adjuvants have been permitted for use with human vaccination. We have previously shown that the administration of naloxone (NLX), a general opioid antagonist, during infection with a non-virulent strain of herpes simplex virus type 1 (HSV-1) could enhance protection against HSV-1 challenge. Here, the adjuvant activity of NLX has been evaluated using a DNA vaccine for HSV-1 as a model. BALB/c mice were divided into four groups; for experimental groups, mice received the glycoprotein D1 (gD1) DNA vaccine alone or in combination with the adjuvant NLX. A positive control group received the KOS strain of HSV-1, and a negative control group received PBS. All mice were immunized three times on days 0, 21 and 42. Three weeks after the last immunization, immune responses against HSV-1 were assessed. Our results indicate that the administration of NLX as an adjuvant increased the ability of the gD1 DNA vaccine to enhance cytolytic T lymphocyte activity, lymphocyte proliferation, delayed-type hypersensitivity and shifting the immune response toward a T helper (Th)1 pattern and improved protective immunity against HSV-1. NLX also increased the IgG2a/IgG1 ratio, though it did not affect the production of HSV-1 antiserum. In conclusion, administration of NLX as an adjuvant in combination with the gD1 DNA vaccine can enhance cell-mediated immunity and shift the immune responses to Th1.

Neuropeptides (SP and CGRP) augment pro-inflammatory cytokine production in HSV-infected macrophages

Neuropeptides are able to modulate cytokine production by macrophages in response to various stimulators. In this study, the effects of neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) on production of pro-inflammatory cytokines TNF and IL-1 beta by macrophages were considered. Mouse peritoneal macrophages were infected with herpes simplex virus type-1 (HSV-1), or remained unstimulated, and cytokine assays were performed after 12 h. IL-1 beta and TNF secretion by unstimulated macrophages have been significantly increased in the presence of SP and CGRP. Each neuropeptide, alone or in coordination with the other, caused significant increase in IL-1 beta and TNF production by HSV-infected mouse peritoneal macrophages. It was concluded that the macrophage-mediated inflammatory response to HSV-1 is enhanced in the presence of these neuropeptides.

Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome.

BACKGROUND/AIMS The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. METHODS The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/75 females, 194 treatment-nai ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. RESULTS In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-nai ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. CONCLUSIONS Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications.

MOLECULAR PHYLOGENETIC ANALYSIS OF IRANIAN HDV COMPLETE GENOME

Hepatitis D (delta) virus (HDV) is a subviral pathogen agent and a satellite of Hepatitis B virus. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, the entire nucleotide sequence of an HDV isolate from an Iranian patient (IR-1) was obtained using twelve pairs of primers to amplify six overlapping fragments covering the whole HDV genome by RT-nested PCR. Phylogenetic and pairwise alignments were done on this new isolate to determine IR-1 position among other isolates. Our results indicate that IR-1 contains 1676 nucleotides encoding 214 a.a. of the hepatitis delta antigen (HDAg). This new isolate belongs to genotype I with most sequence similarity to an Italian HDV isolate (92.6%). At amino acid level, predicted HDAg sequence of IR-1 revealed the most homology with those of Italian and Lebanese isolates. Data analysis confirmed genetic variability and heterogeneity of the HDV species isolated from different geographical areas.

Mutations in the E2 and NS5A regions in patients infected with hepatitis C virus genotype 1a and their correlation with response to treatment

Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF‐2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated‐IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN. J. Med. Virol. 83:1332–1337, 2011.

Characterization of Hepatitis B Virus Genome Variability in Iranian Patients With Chronic Infection, a Nationwide Study

Hepatitis B virus (HBV) isolates from Iranian patients around the country were characterized. Eighty‐one complete genomes from HBV isolates were sequenced and analyzed. The studied population was grouped into three categories including inactive carriers, patients with chronic hepatitis, and patients with liver cirrhosis. Molecular and phylogenetic analyses revealed that Iranian patients were infected with HBV genotype D and subgenotype D1. The most common subtype was ayw2, followed by ayw3 and ayw4. Several deletions and insertions that had no correlation with disease outcome were observed in the HBV genomes. The most frequent mutation in the major hydrophilic region (MHR) of HBV surface antigen (HBsAg) was sP120S. Almost half of the patients studied carried precore (PC) mutant variants and one‐third of the studied population was infected with variants carrying basal core promoter (BCP) mutations. PC and BCP mutations were observed in older patients, especially in those with chronic liver disease. Sixty‐seven patients (82.7%) were HBeAg negative, and the prevalence of precore mutant isolates (G1896A) was higher in this group than in HBeAg‐positive patients. Lamivudine drug resistance mutations were detected after 1 year of treatment in about 30% of lamivudine‐treated patients. In conclusion, these results demonstrate that HBV subgenotype D1 is the only subgenotype circulating in Iran, and there is no evidence of any exotic genotype in the region. The HBV PC (G1896A) mutation may play an important role in the clinical outcome of the disease by increasing the risk of progressive liver disease among Iranian patients infected with HBV. J. Med. Virol. 84:414–423, 2012.

Study of a polymerase chain reaction-based method for detection of herpes simplex virus type 1 DNA among Iranian patients with ocular herpetic keratitis infection

PurposeTo study the presence of the herpes simplex virus type 1 (HSV-1) glycoprotein D gene in tear films of Iranian patients with herpetic keratitis.MethodsTwenty-five tear film and eye swab specimens from 25 herpetic keratitis patients and 10 specimens from 10 healthy volunteers were collected in the Farabi Eye Hospital, Tehran, Iran. HSV-1 DNA was detected by using the nested polymerase chain reaction (nPCR) method. Viral isolation was done using conventional viral techniques. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was used for confirmation of positive cytopathic effect cell culture. The results of a diagnosis by an ophthalmologist team were compared with those of nPCR.ResultsHSV-1 DNA was identified in tear films of 88% (23/25) of suspected herpetic keratitis patients. All healthy controls (100%) had negative PCR results. HSV-1 was isolated in cell culture and confirmed by ELISA in 12% (3/25) of herpetic keratitis patients who had epithelial keratitis. The kappa value showed a high level of agreement between ophthalmologist team diagnosis and the PCR results (kappa = 0.86, P < 0.0001).ConclusionsnPCR is a sensitive, rapid, and powerful tool for detection of HSV-1 DNA in tear films of ocular herpetic keratitis patients and can serve as a supplemental method for diagnosis of herpetic keratitis infection.

Effect of Neuropeptides (SP and CGRP) on Antigen Presentation by Macrophages

There is increasing evidence that neuropeptides regulate various functions of immune cells, including macrophages, which have functional receptors for neuropeptides. We have studied the effects of substance P (SP) and calcitonin gene-related peptide (CGRP) on the presentation of herpes simplex virus type-one (HSV-1) antigens by mouse peritoneal macrophages. Macrophages were treated with live or heat-killed virus in the presence or absence of one or both neuropeptides, then fixed. In vitro proliferation of lymphocytes, derived from intranasally immunized mice, was used to assess antigen presentation by the macrophages. Lymphocytes derived from cervical lymph nodes exhibited a greater proliferative response to heat-killed viral antigens than did lymphocytes derived from spleen. Macrophages treated with live virus did not induce lymphocyte proliferation in the immunized mice, but those treated with heat-killed viral particles did. When macrophages were treated with heat-killed virus in the presence of CGRP, the lymphocyte proliferative response was decreased; however, the effects of SP and SP + CGRP were not statistically significant. The results of interleukin-2 production were in accordance with proliferation assays. These findings suggest that neuropeptides regulate immune responses partly through their effects on macrophage function, including antigen presentation.

Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge.

Influenza virosome is one of the commercially available vaccines that have been used for a number of years. Like other influenza vaccines, the efficacy of the virosomal vaccine is significantly compromised when circulating viruses do not have a good match with vaccine strains due to antigenic drift or less frequent emergence of a pandemic virus. A major advantage of virosome over other influenza vaccine platforms is its intrinsic adjuvant activity and potential carrier capability which have been exploited in this study to broaden vaccine protectivity by incorporating a conserved component of influenza virus in seasonal vaccine formulation. Influenza nucleoprotein (NP)-encoding plasmid was adsorbed onto surface of influenza virosomes as a virosome/DNA vaccine complex. Mice were immunized with a single dose of the influenza virosome attached with the NP plasmid or NP plasmid alone where both influenza virosomes and NP gene were derived from influenza A virus H1N1 New/Caledonia strain. Analysis of the cellular immune responses showed that 5μg (10-fold reduced dose) of the NP plasmid attached to the virosomes induced T cell responses equivalent to those elicited by 50μg of NP plasmid alone as assessed by IFN-γ and granzyme B ELISPOT. Furthermore, the influenza virosome/NP plasmid complex protected mice against intra-subtypic challenge with the mouse adapted H1N1 PR8 virus, while mice immunized with the virosome alone did not survive. Results of hemagglutination inhibition test showed that the observed intra-subtypic cross-protection could not be attributed to neutralizing antibodies. These findings suggest that influenza virosomes could be equipped with an NP-encoding plasmid in a dose-sparing fashion to elicit anti-influenza cytotoxic immune responses and broaden the vaccine coverage against antigenic drift.